JP2015531072A - Stimulated emission suppression microscopy - Google Patents

Abstract

Aberrations in stimulated emission suppression microscopy are corrected using an adaptive optical technique that uses a metric that combines both image sharpness and brightness. The light modulators (22, 32) are used to correct aberrations in one or more of the suppression path (10), the excitation path (12), or the emission path from the sample to the detector.

Description

  The present invention relates to a method of stimulated emission depletion microscopy, computer program code for such a method, and a stimulated emission suppression microscope.

  Stimulated emission suppression (STED) microscopy is used to image not only thin samples such as single cells, but also thick samples such as tissue sections, nematodes, or even living mice. This technique is a fluorescence microscopy technique.

  STED microscopy superimposes a conventional excitation focus with a high intensity second laser focus-suppression focus- that is red-shifted with respect to the peak of the fluorescence spectrum and is characterized by a minimum central intensity (ideally zero intensity). This overcomes the diffraction limit. The suppression laser pushes the excited fluorophore back to the ground state by stimulated emission, thereby suppressing the fluorescence around the excitation focal point, leaving only the fluorescence at the very small minimum center intensity to be imaged. . Increasing the intensity of the suppression beam reduces the effective point spread function (PSF) to a size that is not diffraction limited in principle.

  In practice, the resolution obtained with STED microscopy is strongly dependent on the minimum intensity quality at the center of the suppression focus, and for the minimum intensity that cannot be ignored, the fluorescence is also suppressed at the center of the focus, and the resolution improvement is not , Disabled. Both system-induced and sample-induced aberrations of the suppressed laser beam can dramatically reduce beam quality and create a non-negligible minimum intensity. Therefore, STED microscopy on samples with significant aberrations is not trivial.

  The most commonly used suppression profile created by helical phase masks is highly resistant to beam aberrations, and therefore helical phase masks are widely used in STED, especially for thick samples. However, in a negative aspect, the helical phase mask creates a donut-shaped suppression focus that is confined in the transverse (xy) plane but is still diffraction limited in the axial (z) direction. Resulting in effective PSF. As a result, these STED images are super-resolution in the lateral direction, but not along the optical axis. In order to obtain the most detailed imaging of a tissue's unique three-dimensional configuration, STED microscopy must show an improvement in three-dimensional resolution.

  For thin samples, two three-dimensional STED methods have been demonstrated so far. The highest resolution has been achieved with complex setups that utilize two opposing objective lenses that are incompatible with thick sample shapes. Alternatively, an annular phase filter featuring a center circle with a phase step of π radians (λ / 2) has been applied using a single objective lens. This phase mask creates an annular focus with additional high-intensity protrusions above and below the center minimum, thereby enabling suppression of fluorescence emission in all directions around the center. Unfortunately, this approach is much more susceptible to aberrations than the spiral phase mask approach, and of course, 3D STED imaging has not been demonstrated with thick samples so far.

  One approach to avoiding aberrations is to embed a sample, eg, tissue, in a refractive index matching medium, but is not compatible with a living sample.

  One known technique for STED imaging is E. Auksorius, BR Boruah, C. Dunsby, PM Lanigan, G. Kennedy, MA Neil, and PM French, "Stimulated emission depletion microscopy with a supercontinuum source and fluorescence. This is a technique proposed in lifetime imaging, "Opt. Lett. 33, 113-115 (2008). In this document, a spatial light modulator (SLM) is used to modulate the phase of the STED beam. A hologram is written to the SLM to generate a phase distribution at the +1 diffraction order, and the SLM flexibility is a type I spiral phase distribution or a type II distribution with a central disk phase shifted by π Enable.

  Although this document suggests that aberration correction may be possible using a programmable SLM, the method used refers to a paper containing a direct measurement of focus, ie a direct image of the STED beam focus. To do. This is not feasible for real samples, and for many samples, including biological samples, the aberrations are complex, which creates difficulties for correcting such aberrations.

  Other types of previously published sensorless adaptive optics (AO) microscopes, such as confocal or two-photon microscopes, use simple image quality metrics such as total image luminance (sum of pixel values) doing. The response of these metrics to a properly selected aberration mode provides a well-defined maximum, which is typically quadratic and hence optimization uses a simple algorithm. Sometimes it was done easily.

  However, in a STED microscope, these simple metrics are not very useful. Aberrated STED microscopes do not effectively confine fluorescence in a narrow area, allowing emission from a fluorophore over a larger volume. From this state, if the aberration is corrected, the suppression becomes more effective and there is an inherent decrease in the total image intensity since the effective PSF contains less fluorophore. In other words, the optimal correction cannot be targeted by a simple metric such as image brightness.

  An alternative approach uses a relatively simple adjustment, such as an adjustment of the objective lens correction ring. Such a relatively simple static method cannot correct the complex aberrations actually encountered in STED microscopy.

  A further need is to align the excitation and suppression beams with a STED microscope.

According to a first aspect of the invention,
(A) obtaining a fluorescence image from a stimulated emission suppression microscope having an excitation light path and a first light modulator in the suppression light path;
(B) calculating a metric that combines a measure of image brightness and a measure of image sharpness of the fluorescent image;
(C) adjusting a pattern on the first light modulator;
Repeating (a), (b), and (c) to reduce or reduce optical aberrations by maximizing or minimizing the metric and / or aligning the suppression optical path with the excitation optical path. Including
A method of stimulated emission suppression microscopy is provided wherein the light modulator is a spatial light modulator or a deformable mirror.

  The method according to this aspect uses a novel image quality feedback metric and demonstrates a resolution well below the diffraction limit in all three dimensions when imaging through a sample with optical aberrations such as tissue.

In another aspect,
Forming a synchronized suppression and excitation beam, wherein the suppression beam has a longer wavelength than the excitation beam; and
Directing the excitation beam along the excitation light path through an objective onto the sample to generate fluorescence;
The suppression beam is directed onto a suppression light modulator, and the suppression beam from the suppression light modulator is directed through the objective lens onto the sample to de-excite the fluorescence away from the center. Forming a suppression beam having a point spread function having a minimum value at the center;
Obtaining the fluorescence from the center;
Moving the center relative to the sample to a plurality of positions to construct a fluorescence image;
Determining a metric of the fluorescent image including a measure of image brightness and a measure of image sharpness;
Adjusting the pattern on the suppression light modulator to reduce optical aberrations by maximizing or minimizing the metric;
A method of stimulated emission suppression microscopy is provided.

In another aspect,
(A) obtaining a fluorescence image from a stimulated emission suppression microscope having an excitation light path and a first light modulator in the suppression light path;
(B) repeating (a) using different patterns on the first light modulator to have different enhancement effects;
Combining the fluorescent images;
A method of stimulated emission suppression microscopy is provided.

  Embodiments of the present invention will be disclosed purely by way of example with reference to the accompanying drawings.

1 shows a schematic diagram of an embodiment of the present invention. Fig. 4 shows an image that is corrected using an embodiment of the present invention. Fig. 4 shows an image that is corrected using an embodiment of the present invention. Fig. 4 illustrates an improvement using an adaptive optical technique of an embodiment of the present invention.

  FIG. 1 is a schematic diagram and is not to scale.

  FIG. 1 illustrates an AO STED setup used in an exemplary embodiment. The output from the 80 MHz mode-locked Ti: sapphire laser 2 was passed through the Faraday isolator 4 and the half-wave plate 6 before being split into two beam paths 10 and 12 by the Glan laser polarizer 8. The transmitted beam 10 is used for STED illumination, i.e. for suppression, so the corresponding optical path will be referred to as the suppression pulse beam path 10. This beam is combined into a 19 m long glass block 14, a delay stage 16 for pulse delay adjustment, and an acousto-optic modulator 18 for laser power adjustment before being coupled into a 100m long polarization maintaining single mode fiber 20. Passed through. The glass block and single mode fiber helped to stretch the pulse to hundreds of picoseconds.

  In order to generate a synchronized excitation pulse, the excitation beam along the excitation beam path 12 reflected by the Glan laser polarizer was focused into a photonic crystal fiber 26 (SCG-800, Newport-RTM). The white light spectrum output from this fiber 26 was coupled to an acousto-optic tunable filter 28 for wavelength selection and power control, and then coupled into a second polarization maintaining single mode fiber 30.

  At the output of each single mode fiber 20, 30, the beams 10, 12 are collimated and reflected from respective liquid crystal spatial light modulators 22, 32 (SLM) placed in a plane conjugated to the pupil of the objective lens. It was.

  Half-wave plates 40, 42 in each beam path and quarter-wave plate 44 in the common beam path were used to generate circular polarization of each beam in the sample. In addition, a polarizing beam splitter cube 46 was used in the suppression beam path to clean the beam polarization.

  The beam is shared by a dichroic mirror 50 before being focused by a 100 × / 1.4 NA oil immersion objective 52 (UPLSAPO 100XO / PSF, Olympus) mounted on a commercial microscope stand (IX71, Olympus). Merged into a common path. Sample 56 was attached to an xyz piezoelectric stage 54 (PINano, Physik Instrumente) for scanning. The fluorescence signal is collected by the objective lens, separated from the incident beam path 10, 12 by the dichroic mirrors 48, 50, filtered by the two band pass filters 60, and 62.5 μm core diameter (approximately 0.64 Airy unit). Of 50:50 signal splitting multimode fiber 62. Each output of the multimode fiber 62 was attached to a single photon counting avalanche photodiode 64,66. Image acquisition and instrument control were accomplished using an instrument PC 70 using software 68.

  For imaging, the Ti: sapphire laser 2 was tuned to 770 nm for suppression, and the excitation line at 633 nm was selected from the output of the photonic crystal fiber 26 using AOTF 28. The laser power measured at the objective lens back aperture was 84-132 mW and 0.2-2 μW for suppression and excitation, respectively.

  Images (128 × 128 pixels) were collected at a scan rate of 100 lines / second, a pixel size of 40 nm, and 1-3 frame accumulation (corresponding to 1.28 to 3.84 seconds / image). The line was scanned in one direction using a sinusoidal velocity profile, which resulted in a pixel dwell time of 21.5 μs at the scan center and about twice this value at the end. Therefore, the recorded pixel values were normalized according to the pixel residence time so that the central pixel is divided by 1. Line profile fitting is Lorentz function (for images acquired after aberration correction) or Gaussian function (for images acquired without aberration correction, in which case the observed data fits the Lorentz profile Was not done).

  A STED phase mask as well as aberration correction was realized using an SLM 22 placed in the suppression beam path. The 800 × 600 pixels of the SLM 22 were individually addressed by connecting an SLM as an additional display device to the instrument PC 70.

  Phase modulation from 0 to 2π radians (and more) at the wavelengths used is achieved by changing the gray scale of the phase image supplied to the SLM 22. A phase modulation significantly greater than 2π radians may be created through phase wrapping, thereby converting larger phase values from the range 0 to 2π radians. Although aberrations affect the illumination, emission and suppression beam path, the achievable resolution is dominated by the quality of the suppression focus.

  For this reason, aberration correction using only the SLM 22 in the suppression path 10 is sufficient to greatly improve image quality.

  However, aberrations in the illumination and emission paths are due to fluorescence excitation (by reducing the illumination intensity at the focus), detection efficiency (by blurring the fluorescence focus on the confocal pinhole) and background signal (in the suppression region). The level of fluorescence (externally through excitation and detection). Accordingly, a second SLM 32 was included to provide additional aberration correction in the excitation beam path 12.

  In alternative embodiments, further improvements may be achieved as well with aberration correction in the emission path.

  Instead of using direct reflection at the SLM 22, 32, an off-axis hologram is used, which directs a non-zero order diffracted beam, here the primary beam, forward through the objective lens. Use of such a diffracted beam is beneficial because it separates phase modulated light from any directly reflected unmodulated light.

  In the described arrangement, the active pupil creates an SLM22 by creating such an off-axis hologram (FIG. 1 inset) —a circular-shaped blazed grating that diffracts light into the first diffraction order away from the main reflection axis. As defined above, the diffracted light was focused into the sample. The remaining non-diffracted fraction of the incident light (as well as the light reflected from the outer part of the active pupil) is simply reflected along the main reflection axis, about 3 μm away from the first focus, and much more than the suppression spot A weak second focus was formed in the sample. This approach ensured that any residual light resulting from imperfect phase modulation could not interfere with the desired suppression focus and degrade its quality.

  We note that non-diffracted light can also be blocked at an intermediate focal plane if necessary. The phase mask for creating the suppression focus was generated by adding a corresponding function to the SLM phase pattern. In this way, a phase mask may be added, modified and replaced fairly easily, and compared to a system in which the manufactured phase plate is physically placed in the optical path.

  This microscope used an oil immersion lens 52 having a numerical aperture (NA) of 1.4. The specimens were mounted in an aqueous medium (phosphate buffered saline, PBS) with a refractive index n = 1.34 or glycerol (n = 1.47). For aqueous encapsulants, the critical system NA is limited to the encapsulant's refractive index due to total internal reflection at higher angles of incidence of the focused beam. We therefore limited the active aperture defined on the SLM to a size corresponding to an objective lens NA of 1.325. This allowed us to effectively mimic the performance of a water immersion objective with a NA = 1.325 higher than a commercial objective.

  The SLM enables adaptive aberration correction through the addition of additional phase functions to the SLM. In the system described here, we used a sensorless AO configuration to determine the aberrations induced by the system and the specimen. In sensorless AO, the aberration is estimated through analysis of a set of images obtained using a series of predetermined bias aberrations. This process requires 1) extension of aberrations using an appropriate set of modes, and 2) definition of image quality metrics that are maximized to find optimal aberration correction.

  Aberrations were expressed using a Zernike polynomial expansion modified to remove small image shifts introduced by displacement (tip, tilt, defocus) modes. . Table 1 lists the Zernike modes (according to conventional numbering) used for aberration correction in this work. Zernike polynomial modes are widely used to model aberrations in adaptive optics. One reason for this is the orthogonality between modes, which is higher order of focal displacement (due to tip, tilt and defocus modes) and focal distortion (astigmatism, coma and spherical aberration). Have useful practical results, such as separation (through mode). However, this separation of effects is only true for a paraxial approximation and a uniform pupil function.

  Those skilled in the art will note that alternative modes may be used to represent aberrations and that the method will function normally in such alternative modes.

These approximations are no longer valid when using phase masks and high NA lenses in STED microscopy. We have experimentally observed that the zero intensity point of the suppression focus may shift laterally, for example when Zernikecoma aberration is applied using an SLM. A similar axis shift was observed when applying the Zernike spherical aberration mode. This behavior has been confirmed through theoretical modeling (data not shown). To eliminate these displacement effects, we measured the focus shift induced for each Zernike mode using images of golden beads. Using a linear approximation of zero shift, each applied Zernike mode to ensure that the proportional amount of tip, tilt, or defocus mode (as appropriate) does not shift the intensity zero point position. Added to This procedure defined a modified set of “displacement-free” Zernike modes that were used in subsequent experiments. Subsequent references to Zernike mode will now refer to the Zernike mode without displacement.

  We have observed that near the optimal correction, adjustment of one aberration mode caused a decrease in image intensity, while others led to an increase (FIG. 2). Therefore, the luminance metric alone is inappropriate for a STED microscope.

  Metrics related to image sharpness may seem more suitable in principle, but optimization based purely on this is deceived by background noise (noise is imaged) It tends to be “sharper” than the specimen) and is therefore susceptible to degradation of the STED focus. Furthermore, it has been found that the sharpness metric response to several aberration modes is very flat near the optimal correction and shows a change close to the fourth order rather than the second order.

  In practice, the property we want from a STED image is high brightness combined with high resolution. The metric used attempts to optimize both image brightness and image sharpness with a combined approach, which succeeds when either individual approach can fail.

In one embodiment, the combination metric is
Where B and S are the luminance and sharpness metrics, respectively. The expression in parentheses in Equation 1 is effectively a luminance metric only when the sharpness metric S is above a certain threshold S _T (typically selected to be 90% of peak sharpness). A logistic function that introduces the contribution of B. Parameter β is empirically chosen to balance the contributions from S and B, the parameter k has been used to adjust the rate of transition logistic function in the region of the S ≒ S _T. The constant σ was +1 or −1 depending on whether the aberration mode in question produced a minimum or maximum at B for a magnitude of zero aberration.

More generally speaking, the metric M combines sharpness and brightness. In an embodiment, the metric M is alternatively
Where f (S) is a function of S having a higher absolute value for S above S _T and a lower absolute value for S below S _T.

  Image luminance metric B was calculated as the sum of pixel values in the confocal or STED image. This image luminance metric B was used solely as a metric for adjustment of the excitation beam path 12, ie as a second metric B used for aberration correction using the SLM 32 in the excitation beam path.

The sharpness metric S is defined as the second moment of the image Fourier transform (FT),
Where I ^ _{n, m} is the discrete FT of the image, n ′ = n− (N−1) / 2, m ′ = m− (M−1) / 2, and n and m is a coordinate ranging from 0 to N-1 or M-1. The function μ _{n, m} is
Where w is the radius (in pixels of the image FT) defined as the size of the field of view divided by the upper limit to the expected resolution (eg 200 nm).

  This mask is used to block higher spatial frequencies that would otherwise cause S to be dominated by noise. The motivation for this choice of metric is the inverse correlation between the width of the image feature and the width of the corresponding spatial frequency spectrum of the image, and a sharp image leads to a wide image FT. Since the second moment calculation of Equation 3 is a measure of the width of the image FT, a large value of S results in a sharp image resulting from a narrow PSF. The definition of M should mean that it is readily applicable to any sample characterized by a structure that is smaller than the diffraction limit.

  Note that when imaging with a phase mask centered at λ / 2, the second and third spherical aberration modes lead to a decrease in image brightness, while the first spherical aberration mode leads to an increase in brightness. is important. These results indicate that the objective correction ring adjusts the combination of these spherical aberration modes, so manual adjustment of the objective correction ring to maximize image brightness is not necessarily applied to 3D STED microscopy. Indicates that it may be.

In the sensorless adaptive optical configuration of this embodiment, the image (single accumulation) is confocal (for excitation path correction) or STED (for suppression path correction) with the addition of bias aberration bZ ' _i for a given mode Z' _i ) Mode, where b was an appropriately selected bias amplitude. Typically b was chosen to range from -1 to +1 radians while collecting multiple images for each Zi. For each image, the appropriate metric was calculated (B for excitation path correction, M for suppression path correction) and then plotted as a function of bias amplitude b. The corrected aberration a _corr was then estimated as the peak of the curve calculated by fitting a quadratic function to the data. If the quadratic approximation did not represent data, the peaks could be manually identified.

The aberration a _corr Z ′ _i was then added to the SLM phase pattern, and this measurement and correction cycle was then repeated for each mode of interest.

  Aberrations due to coma, astigmatism, and trefoil were sometimes evaluated using a single iteration of our correction routine. The spherical aberration mode is typically the dominant aberration in the experiments performed here and typically required 1-4 correction iterations to converge to the optimal value. However, when the results for correcting the excitation beam path are used as a starting point for correction of the suppression beam path, one or two iterations are typically sufficient to evaluate spherical aberration. Met. All AO STED images shown have the same field of view used to perform aberration correction.

  To verify the correction procedure using aberrations encountered in biological specimens, 200 nm crimson beads were added to the top surface of a zebrafish retinal section attached to a # 1.5 cover glass and then on a microscope slide Encapsulated in PBS (to mimic the aqueous embedding conditions of live sample imaging). 2A-L show xy and xz images of individual beads after focusing through a retinal section to about 14 μm or about 25 μm.

  Images A, D, G, J, and M show a confocal image, i.e., no suppression beam path is used. Images B, E, H, K, and N show the improvement in resolution using STED, i.e., using a suppression beam path. Images C, F, I, L, and O show an improvement using aberration correction in the suppression beam path 10 using the SLM 22.

  In this example, all aberration corrections were made only on the suppression beam path 10 in order to demonstrate that correction of the suppression beam path is possible without prior knowledge of the corrections made to the excitation path 10. . These data also demonstrate that the quality of the suppression focus is the dominant factor in achieving sub-diffraction resolution in STED images. Although relatively few aberration mode corrections were sufficient to obtain axial resolutions of about 200 nm and about 250 nm, respectively, for imaging depths of about 14 μm and about 25 μm, we found that higher order aberrations We expect that including the correction routine in the correction routine should further improve the STED resolution. 2M-O show a volume rendering of the data depicted in FIGS. 2A-F and represent the first three-dimensional super-resolution STED image recorded through the scattering tissue.

  In order to demonstrate the correction of aberrations induced by a highly aberrant sample, using both beam paths 10, 12, we also fluoresced through a layer of glycerol (n = 1.47). The beads were also imaged. Samples with fluorescent beads were prepared by attaching 100 nm crimson beads to a microscope slide coated with polyerlysine, and the beads were covered with # 1.5 coverslips and about 55 μm glycerol used as embedding agent. Imaged through.

  FIG. 3 shows bead images before and after aberration correction in the normal confocal imaging mode and the STED imaging mode. In FIG. 3, image A shows an image in confocal mode without STED, and image B has correction for sample-induced aberrations only in the excitation beam path 12 using the SLM 32 and the second metric B. , STED shows image A not yet present. Image C shows an image that uses STED, but only uses SLM 32 to correct excitation beam path 12, ie image B, but with STED. Image D shows the result of correcting aberrations in both the suppression beam path 10 and the excitation beam path 12.

  The aberration correction routine began with correcting the excitation beam path 12 by adjusting the SLM 32 using only the image brightness B as a measurement reference in the confocal imaging mode.

  The resulting correction value was then used as a starting point for correction of the suppression beam path 10 using the combined metric defined in Equation 1 in STED imaging mode. 3A and B illustrate the benefits of using a second SLM to correct aberrations in the excitation beam path (for confocal imaging). As shown in FIG. 3C, the uncorrected STED image shows a modestly improved resolution compared to the confocal image, but the non-zero center intensity suppresses fluorescence, thus significantly reducing the signal level. At the expense of On the other hand, aberration correction significantly improves both the intensity and resolution of the STED image (FIG. 3D).

  The axial profiles of STED and AO STED images are plotted for comparison (FIG. 4), and the results show an approximately 5-fold increase in peak signal as well as an approximately 3.2-fold improvement in resolution.

  We demonstrate that the STED images shown in FIGS. 2 and 3 before and after aberration correction were images of the same bead on which the correction routine was performed, and therefore photobleaching was not important in these experiments. Keep in mind that Labeling conditions in biomedical imaging applications are likely to use a fluorophore with low photostability, which may not allow the collection of 5-7 STED images per aberration mode in some cases. However, it has been demonstrated that only 2N + 1 images are needed to correct the N aberration modes, and we expect this approach to be compatible with the method presented here. In addition, applications that would otherwise be suppressed by photobleaching may benefit from implementing triplet state relaxation during STED imaging by increasing the scan speed or using a lower repetition rate laser.

  The proposed setup may also be used for automatic alignment. In particular, in order to align the excitation path with the suppression path, the pattern on the suppression path path modulator adds a certain amount of beam mode, such as tip or tilt, to the light modulator and then collects the image. It is adjusted by. Appropriate metrics are calculated and optimized to align the excitation and suppression paths.

  Just as used for aberration correction, the correct choice of metric is important in the case of automatic beam alignment. Near optimal alignment, the STED image will be maximally bright when the zero intensity position is aligned to the center of the excitation focus Gaussian focus. Unfortunately, away from optimal alignment, the non-overlapping beams yield a conventional confocal image that is also bright. Accordingly, a simple metric based on luminance is not appropriate.

  Therefore, a metric M that combines brightness and sharpness is used in the coarse alignment step to correct for beam misalignment. This is followed by a fine alignment step using only the metric B related to luminance.

  A particular benefit of the proposed approach is that both optical aberration correction and coarse alignment can use the metric M, and so can be performed as a single operation, ie a single optimization approach Note that it is possible to align and correct aberrations.

  In the paper by Auksorius referenced above, it was reported that the SLM was used to correct system aberrations in the suppression beam path through direct observation of the suppression focus. However, this method is not useful for correction of aberrations introduced in actual specimen microscopy, where one can usually only access the fluorescent image. The present invention allows aberration correction using the super-resolution image itself, which is an important step towards making automated AO useful for in vivo super-resolution microscopy.

  SLM provides a convenient way to combine a STED phase mask and adaptive aberration correction in the same microscope.

  One skilled in the art will recognize that changes may be made to the embodiments described above.

  The embodiments described above use SLMs in both the suppression and excitation paths. In some embodiments, the SLM is provided only on the suppression path. In other embodiments, the light modulator may be provided in the fluorescence emission and detection path between the sample and the detector.

  There are alternatives to the use of SLM as an optical modulator. For example, a deformable mirror may be used instead of an SLM as a light modulator. An alternative approach using a deformable mirror would have the advantage that all three (suppression, excitation, and emission) beam paths can be corrected with the same device. However, unlike SLM, the continuous reflective surface of the mirror device does not allow the generation of a STED phase mask that requires discontinuous phase jumps.

  A further approach is to use an optical modulator where the beam paths coincide to allow multiple beam paths to be corrected using a single optical modulator.

  An alternative embodiment illustrating these options is FIG. In this embodiment, there are two light modulators. One of these is the suppression beam path SLM 22 installed only in the suppression path, and the second optical modulator is the combined beam path deformable mirror 102 installed where the three beam paths coincide. Note that the SLM 32 in the excitation path in FIG. 1 is replaced with a simple mirror 100.

  In this approach, the suppression beam path SLM 22 is used to correct the phase pattern to shape the suppression focus and provide aberration correction. The second light modulator, i.e. the deformable mirror 102, is used to correct each of the suppression, excitation and emission paths.

  One skilled in the art will recognize that a light modulator may be provided in each of the suppression, excitation, and emission paths or may be shared as in the embodiment of FIG.

  Alternative sample mounting arrangements and methods of arranging optical components to direct light along their respective beam paths may be used as is known in the art.

2 Ti: sapphire laser 4 Faraday isolator 6 Half-wave plate 8 Glan laser polarizer 10 Transmitted beam, suppression beam path, suppression path 12 Excitation beam path, excitation path 14 Glass block 16 Delay stage 18 Acoustooptic modulator 20 Polarization maintaining single Mode fiber 22 Spatial light modulator, SLM
26 Photonic crystal fiber 28 Acousto-optic tunable filter, AOTF
30 polarization-maintaining single mode fiber 32 spatial light modulator, SLM
40 half-wave plate 42 half-wave plate 44 quarter-wave plate 46 polarizing beam splitter cube 48 dichroic mirror 50 dichroic mirror 52 oil immersion objective lens 54 piezoelectric stage 56 sample 60 bandpass filter 62 multimode fiber 64 avalanche photodiode 66 avalanche Photodiode 68 Software 70 Instrument PC
100 Simple mirror 102 Deformable mirror

Claims (24)

A method of stimulated emission suppression microscopy using adaptive optics, comprising:
(A) obtaining a fluorescence image from a stimulated emission suppression microscope having an excitation light path and a first light modulator in the suppression light path;
(B) calculating a metric that combines an image brightness measure and an image sharpness measure of the fluorescent image;
(C) adjusting a pattern on the first light modulator;
Repeating (a), (b), and (c) to reduce or reduce optical aberrations by maximizing or minimizing the metric and / or aligning the suppression optical path with the excitation optical path. Including
The method of stimulated emission suppression microscopy, wherein the first light modulator is a spatial light modulator or a deformable mirror. (A) acquiring the fluorescent image comprises:
Forming a synchronized suppression and excitation beam, wherein the suppression beam has a longer wavelength than the excitation beam; and
Directing the excitation beam along the excitation light path through the objective lens onto the sample to generate fluorescence;
The suppression beam is directed along the suppression optical path onto the suppression light modulator, the suppression beam from the suppression light modulator is directed onto the sample through the objective lens, and the fluorescence away from the center is deexcited. Forming a suppression beam having a point spread function having a minimum value at the center,
Obtaining the fluorescence from the center;
The method of stimulated emission suppression microscopy according to claim 1, comprising constructing the fluorescence image by moving the center relative to the sample to a plurality of positions. The method further comprises:
Directing the excitation beam onto an excitation light modulator and directing the excitation beam from the excitation light modulator through the objective lens onto the sample to generate fluorescence;
3. The method of claim 2, comprising adapting a pattern on the excitation light modulator to reduce optical aberrations by maximizing or minimizing a second metric. The path from the sample to the detector is an emission optical path, and the method further comprises:
Directing the light from the sample through the objective lens onto a second light modulator and then onto the detector;
4. A method according to claim 2 or 3, comprising adapting a pattern on the second light modulator to reduce optical aberrations by maximizing or minimizing further metrics. The second light modulator is also in the excitation light path;
Adapting the pattern on the second light modulator to reduce optical aberrations by maximizing or minimizing further metrics is an optical aberration in both the excitation and emission optical paths. 5. The method of claim 4, comprising adapting the pattern on the second light modulator to reduce. The second optical modulator is in the suppression optical path in addition to the emission optical path and the excitation optical path;
Adapting the pattern on the second light modulator to reduce optical aberrations by maximizing or minimizing a further metric may include the suppression optical path, the excitation optical path, and the emission optical path. 6. The method of claim 5, comprising adapting the pattern on the second light modulator to reduce optical aberrations at each.   The method according to any one of claims 1 to 6, comprising correcting a plurality of optical aberration modes.   The pattern on the suppression light modulator is a pattern configured to direct a non-zero order diffraction peak on the sample as the suppression beam having a point spread function. The method described in 1.   9. The metric according to any one of claims 1 to 8, wherein the metric combines a sharpness measure that measures image sharpness with a luminance measure, the luminance measure being reduced when the sharpness measure is lower. the method of. The metric M is
Where M is the metric, S is the image sharpness scale, B is the image brightness scale, and σ is the minimum brightness that the corrected aberration mode produces maximum brightness. Is a constant having a value of -1 or 1, depending on whether or not, β is a constant, and f (S) has a larger absolute value for values of S above the threshold S _T and said threshold it is a function of S having a smaller absolute value for values of S below S _T, the method according to any one of claims 1 to 9. The metric M is
The method of claim 10, wherein k is a proportionality constant.   12. A method according to any one of the preceding claims, wherein the measure B of image brightness is calculated as the sum of pixel values in the image. Said measure S of image sharpness is defined as the second moment of the image Fourier transform (FT),
Where I ^ _{n, m} is the discrete FT of the image, n ′ = n− (N−1) / 2, m ′ = m− (M−1) / 2, and n and m Are coordinates ranging from 0 to N−1 or M−1, respectively, and the function μ _{n, m} is
The method according to claim 1, which is a circular mask defined by: (D) obtaining a fluorescence image from the stimulated emission suppression microscope;
(E) calculating a metric for measuring image brightness, image sharpness, or both of the fluorescent image;
(F) adjusting a pattern on the light modulator;
Further comprising performing a fine alignment procedure to provide one or more fine alignments of the suppression light beam path, the excitation light beam path, and the emission beam path by repeating The method according to any one of 1 to 13.   15. A method according to any one of claims 1 to 14, wherein the light modulator is a deformable mirror. (A) obtaining a fluorescence image from a stimulated emission suppression microscope having an excitation beam along the excitation path and a spatial light modulator in the suppression optical path of the suppression beam;
(B) calculating a metric combining a measure of image brightness and a measure of image sharpness of the fluorescent image;
(C) adjusting the pattern on the spatial light modulator;
Repeating (a), (b), and (c) to reduce optical aberrations by maximizing or minimizing the metric or to align the suppression beam and the excitation beam;
A computer program product configured as follows: The acquired image is configured to be acquired from the stimulated emission suppression microscope, and the microscope further includes a second spatial light modulator;
The computer program product of claim 16, wherein the computer program product is configured to adjust a pattern on both the first and second spatial light modulators. The metric M is
Where M is the metric, S is the image sharpness scale, B is the image brightness scale, and σ is the minimum brightness that the corrected aberration mode produces maximum brightness. Is a constant having a value of -1 or 1, depending on whether or not, β is a constant, and f (S) has a larger absolute value for values of S above the threshold S _T and said threshold is a function of S having a smaller absolute value for values of S below S _T, a computer program product according to claim 16 or 17. Said measure S of image sharpness is defined as the second moment of the image Fourier transform (FT),
Where I ^ _{n, m} is the discrete FT of the image, n ′ = n− (N−1) / 2, m ′ = m− (M−1) / 2, and n and m Are coordinates ranging from 0 to N−1 or M−1, respectively, and the function μ _{n, m} is
The computer program product of claim 18, wherein the computer program product is a circular mask defined by: A light source;
A splitter for dividing light from the light source into a suppression light path and an excitation light path;
An objective lens that directs the light passing through the excitation light path onto a target;
A frequency shifter that shifts the frequency of light traveling through the suppression light path to a lower frequency;
An optical modulator configured to direct the light traveling through the suppression optical path through the objective lens onto the sample with a point spread function having a minimum value at the center so as to de-excite fluorescence away from the center When,
A sensor for acquiring fluorescent light from the center;
Optical aberrations by constructing a fluorescence image of the sample from light detected at multiple centers, measuring a metric including a measure of image brightness and a measure of image sharpness, and maximizing or minimizing the metric Or adapting a pattern on the light modulator to align the suppression light path and the excitation light path;
A stimulated emission suppressing microscope.   The stimulated emission suppression microscope according to claim 20, further comprising a second light modulator in the excitation light path.   The second optical modulator is a deformable mirror in each of the suppression optical path, the excitation optical path, and the emission optical path, and the emission optical path is an optical path between the sample and the sensor. The stimulated emission-suppressing microscope according to 21.   23. The stimulated emission of claim 20, 21, or 22, wherein the first light modulator is configured to display a diffraction pattern that directs a non-zero order diffraction peak with a point spread function on the sample. Suppression microscope. The metric M is
Where M is the metric, S is the image sharpness scale, B is the image brightness scale, and σ is the minimum brightness that the corrected aberration mode produces maximum brightness. Is a constant having a value of -1 or 1, depending on whether or not, β is a constant, and f (S) has a larger absolute value for values of S above the threshold S _T and said threshold for values of S below S _T is a function of S having a smaller absolute value, stimulated emission suppression microscope according to claim 20, 21, 22, or 23.